<B>Molecular Genetic Study of Stem Cell Regulation and Animal Aging in Drosophila and Mice</B> Tissues and organs in our body are generated and maintained by stem cells. Stem cells possess unlimited self-renewal potency. Through asymmetric cell division, a stem cell in adult tissues can produce an offspring that will maintain the stem cell populations and a daughter cell that will differentiate into various short-lived cell types to replace damaged or dying cells. Tumors may originate from a few transformed cancer stem cells. Stem cells have immense potential for therapeutic use in regenerative medicine and for developing anticancer therapies that specifically eliminate cancer stem cells. To make use of this potential, we must first understand the molecular parameters that define a stem cell and the mechanisms that regulate stem cell behavior. Many human diseases, such as neurodegenerative diseases and cancers, are age-related. However, aging is still a fundamental, unsolved mystery in biology. My laboratory is studying stem cell regulation and animal aging in Drosophila and mouse in vivo model systems. Our current projects on stem cell regulation and animal aging are derived from our early studies in the JAK/STAT and JNK/JUN signal transduction pathways in Drosophila. The JAK/STAT signal transduction pathway regulates germline stem cell self-renewal in the Drosophila testis, while the JNK/JUN signal transduction pathway regulates stress response and lifespan in the fly. Consequently, we have begun to study the molecular mechanisms whereby these two signaling pathways regulate stem-cell self-renewal and aging in Drosophila. The following sections briefly describe our recent accomplishments and ongoing studies in this area. <b>I. Signalings that regulate male germline stem cell self-renewal or differentiation</b> Drosophila testis offered an excellent genetic system for studying stem cell regulation at the molecular and cellular level. At the tip of the Drosophila testis (apex) is a germinal proliferation center, which contains the germline and somatic stem cells that maintain spermatogenesis. Each germline stem cell (GSC) is encysted by two somatic stem cells (SSCs). Both GSCs and SSCs anchor to a group of 12 nondividing somatic cells, called the hub, through cell adhesion molecules. The hub defines the stem-cell niche by expressing the growth factor Unpaired (Upd), which activates the JAK/STAT pathway in GSCs to regulate stem-cell self-renewal. We have undertaken a number of genetic experiments in the last two years to further understand stem cell regulation and the role of the JAK/STAT signaling in this system. We have focused on (1) identifying the signal (s) that regulates stem cell anchoring to the niche, (2) identifying the downstream targets of the JAK/STAT signal transduction pathway, and (3) understanding how the GSCs and SSCs coordinate their self-renewal and differentiation. We have made significant progress on each of these topics as described below. (1) In a genetic screen for mutations that interact with the JAK/STAT signal transduction pathway in regulating male GSC fates, we identified a small GTPase Rap guanine nucleotide exchange factor (Gef26) from our library of P-element mutations. The protein has a PDZ domain, a Ras-binding domain, a cAMP/cGMP-binding domain, and a Rap-binding domain. Mutations of Gef26 cause loss of both GSCs and SSCs in the fly testis. We demonstrated that Rap-GEF/Rap signaling controls stem cell anchoring to the niche through regulating DE-cadherinmediated cell adhesion. The Gef26 mutation specifically impairs adherens junctions at the hub-stem cell interface, which results in the stem cells drifting away from the niche and losing stem cell identity (Wang et al., 2006). (2) We recently identified that the Drosophila ortholog of the human kidney tumor suppressor BHD regulates GSC maintenance and functions downstream of the JAK/STAT and Dpp signal transduction pathways (Singh et al., 2006). (3) In many tissues, two or more types of stem cells share a niche, and how the stem cells coordinate their self-renewal and differentiation is poorly understood. In the Drosophila testis, germline stem cells (GSCs) and somatic stem cells (SSCs) contact each other and share a niche (the hub). The hub expresses a growth factor Unpaired (Upd) that activates the JAK/STAT pathway in GSCs to regulate the stem cell self-renewal. However, it is not clear how SSC cells are regulated and how SSCs and GSCs coordinate their self-renewal or differentiation. We recently have identified the signaling that regulates SSC self-renewal and also discovered how SSCs and GSCs coordinate their self-renewal or differentiation (Zheng et al., submitted). Our recent genetic screens have identified a number of mutants that either suppress or enhance the stem cell phenotypes of mutations in Gef26 or in the JAK/STAT signal transduction pathway. We also identified a number of proteins that bind Gef26 in a yeast two-hybrid screen. Further characterizing these new mutants and Gef26-binding proteins in next few years will provide new insights into molecular mechanisms governing stem cell anchoring, self-renewal, and stem cell-stem cell coordination. <b>II. The Adult Drosophila Malpighian Tubules Are Maintained by Multipotent Stem Cells </b> All animals must maintain a constant body composition by excreting the waste products of metabolism. Excretion is performed by the kidney in vertebrates and by the Malpighian tubules in Drosophila. The mammalian kidney has an inherent ability for recovery and regeneration following ischemic injury. Stem cells and progenitor cells have been proposed to be responsible for repair and regeneration of injured renal tissue. In Drosophila, the Malpighian tubules are thought to be very stable, and no stem cells have been described in the organ. We have recently identified pluripotent stem cells in the region of lower tubules and ureters of the Malpighian tubules. Using lineage tracing and molecular marker labeling, we demonstrated that several differentiated cells in the Malpighian tubules arise from the stem cells and an autocine JAK-STAT signaling regulates the stem cells self-renewal. Identifying adult kidney stem cells in Drosophila may provide important clues for understanding mammalian kidney repair and regeneration during injury. This work was recently published on Cell Stem Cell as a featured article (Singh et al., 2007). <b>III. Signalings that regulate the fly lifespan</b> We screened P-element mutants, either generated by us or obtained from public stock centers, and identified 40 long-lived mutants. We are currently exploring the molecular mechanism of how these new mutants and the JNK signaling pathway cooperatively regulate fly lifespan. <b>IV. Stem cell regulation and animal aging in mice</b> We are using knowledge gained from the Drosophila systems as guiding principles for studying stem cell regulation and animal aging in the mouse system. We are knocking out the corresponding mice orthologs of genes that play important roles in stem cell regulation and animal aging in the Drosophila systems. We are also developing cell labeling systems to specifically label stem cells and cancer stems cells in the mouse system. Our goal on this project is to first develop a labeling system that allow us to identify stem cells and then study stem cell regulation through manipulating gene activity in mice. The knowledge gained from our research will form the scientific foundation for developing anticancer therapies that specifically eliminate cancer stem cells